Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
Uploaded: 2018-05-31T13:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

The protocols below refer to the isolation of mouse MoDC, yet the overall principles may apply to other DC subsets cells, as well. 12 - 16-week-old C57Bl/6j mice were maintained in an American Association for the Accreditation of Laboratory Animal Care&#8211;accredited animal facility. All protocols were approved by Stanford University and Tel-Aviv University Institutional Animal Care and Use Committee.
1. Isolation of Tumor Associated Monocyte-Derived DC
<ol>
	<li>In a laminar flow hood, remove tumors from CO2 euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS). 
	NOTE: It is highly recommended to shave the mice and spray them with 70% ethanol before removing the tumors, to minimize potential contaminations from the fur. Be certain that the tumor does not exceed 25 - 40 mm2 since larger tumors have fewer DC that tend to be fragile and prone to undergo cell death. If larger numbers of DC are needed, each mouse can be injected with tumors cells at multiple sites.</li>
	<li>Under a sterile laminar hood, chop the tumors using surgery scissors into small pieces (approximately 1 x 1 mm2).
	<ol>
		<li>Add all tumor fragments from one mouse into a 30-mL sterile flat-bottom tube with magnetic stir bars, which contains 5 mL of Hank&#39;s balanced salt solution (HBSS), 2 mg/mL collagenase IV, and 0.01 mg/mL DNase I. 
		Caution: Myeloid cells produce energy through glycosylation, even under steady state. Therefore, only use media that contains glucose (e.g. HBSS, RPMI, and DMEM), as glucose starvation for as little as 10 - 15 min will result in cell death and apoptosis within 24 h. Use only a low endotoxin collagenase IV, as collagenase is produced by Gram-positive bacteria (Clostridium histolyticum).</li>
	</ol>
	</li>
	<li>Stir at about 200 - 400 rpm in a 37 oC incubator with a magnetic stirrer for 20-30 min.</li>
	<li>Add 5 mL of complete media and re-suspend vigorously.</li>
	<li>Filter cells through a 70-&#181;m cell strainer. Centrifuge at 4 oC 400 rcf for 5 - 10 min to pellet the cells. 
	Caution: Do not attempt to isolate the cells directly following enzymatic digestion, as some tumors are necrotic and contain relatively large amounts of cell debris or extracellular matrix. Apply cells on 15% density gradient medium to obtain only the cells.</li>
	<li>Suspend 9 mL of density gradient medium with 1 mL of 10x PBS to adjust osmolality, and to obtain 100% Percoll stock solution. Mix 1.5 mL density gradient medium stock solution with 8.5 mL HBSS and mix it vigorously with tumor-derived cell pellet. Gently layer 2 mL of DMEM on the top of 15% density gradient medium and centrifuge at 400 rcf for 20 min at room temperature. Discard the supernatants, as the cells will form a pellet at the bottom of the tube.
	<ol>
		<li>Wash the pelleted cells twice by re-suspending them in 10 mL HBSS containing 2% FBS, 1% penicillin/streptomycin, and 10 mM EDTA (Isolation Buffer), and centrifuge at 4 oC 400 rcf for 5-10 min to pellet the cells.</li>
		<li>Count cells on a hemocytometer using trypan blue under a light microscope.</li>
	</ol>
	</li>
	<li>Re-suspend 1 x 108 cells in 1 mL isolation buffer and incubate them at 4 oC with 30 &#956;L of CD11b+ conjugated magnetic beads for 15 min. 
	Caution: Avoid the use of CD11c-conjugated beads, as this will activate the DC and induce cell death. Do not attempt to block Fc&#947;RII and Fc&#947;RIII with anti-CD16/32 antibodies. Blocking antibodies ligate Fc&#947;R and induce strong phosphorylation of MAP kinases and prime the DC.
	<ol>
		<li>Remove the excess unbound beads by adding 9 mL of isolation buffer and centrifuge cells at 400 rcf for 5 - 10 min at 4oC.</li>
	</ol>
	</li>
	<li>Aspirate the supernatant and re-suspend cells in 1 mL isolation buffer. Apply the cells on a prewashed magnetic column. Wash the column twice with 3 mL of isolation buffer.
	<ol>
		<li>Remove the column from the magnet. Pipette 6 mL of isolation buffer onto the column and flush out the magnetically-labeled cells into a sterile collection tube by pushing the plunger. Centrifuge cells at 400 rcf for 5 - 10 min at 4 oC.</li>
		<li>Re-suspend cells at 100 &#956;L per 1 x 107 cells and stain with the following fluorophore-conjugated antibodies: Linage negative (TCRb, Siglec F, B220, CD19, FceRI&#160;and Ly6G),&#160;MHCII, Ly-6C.</li>
	</ol>
	</li>
	<li>Sort cells by gating the small cells, using side scatter (SSC) and forward scatter (FSC) and culture in complete medium supplemented with 5 ng/mL GM-CSF. Incubate cells for 1 hour at 3 7&#160;oC in order to allow macrophages to adhere the plate. Then, transfer the loosely and non-adherent cells to a new culture dish. 
	NOTE: Mouse MoDC are defined as CD11b+/CD11c+/MHCIIhi/Ly6Clo/int 7,26,27. Expression of Ly6C may vary dramatically between tumor models, and in some models (e.g. LMP) MoDCs completely lack Ly6C expression. Overall, one 100 mm3 B16F10 tumor typically contains 5 x 104 of DC, resulting in approximately 4 - 5 x 106 total cells. Of these cells, 10 - 15% are immune cells and 8 - 10% are MoDC. Increasing the DC numbers can be achieved by injecting mice with tumors at multiple sites. Sort only small SSC/FCS cells, as other myeloid cells can express markers such as CD11c and Ly6C.&#160; 
	Optional: Sort the tumor-infiltrating monocyte as small SSC/FSC cells that express Ly6Chi and are negative for MHCII. Afterward, culture monocytes in vitro with 50 ng/mL GM-CSF to obtain DC.</li>
</ol>
2. Isolation of Monocyte-derived DC from Peripheral Blood
NOTE: Since mature MoDCs are relatively rare in mouse blood, the protocol below refers to their derivation in vitro from sorted monocytes.
<ol>
	<li>To increase the levels of monocytes in blood, anesthetize mice with 4% isoflurane and inject them subcutaneously (s.c.) with 1 &#956;g of GM-CSF in 50 &#956;L PBS.</li>
	<li>After 1-2 h, sacrifice mice using CO2. 
	Caution: Do not sacrifice mice by cervical dislocation, as this will cause internal bleeding.</li>
	<li>Immediately after euthanization,spray the mouse with 70% ethanol and remove the skin that covers the heart using surgical scissors, under a sterile laminar flow hood. Clean the scissors with ethanol and cut the right atrium of the heart.Slowly, flush the heart through the right ventricle with 20 mM EDTA HBSS using a 10-mL syringe and 25-G needle.
	<ol>
		<li>Collect the blood with a sterile syringe from the pleural cavity into a sterile tube containing heparan sulfate and EDTA. Continue to flush the heart until the liver and lungs become pink or white.</li>
	</ol>
	</li>
	<li>Apply blood onto a density gradient mediumand centrifuge at 400 rcf at room temperature for 15 min with low brake. 
	Caution: Use endotoxin-free density gradient medium (usually less than 0.12 EU per mL).
	<ol>
		<li>Collect mononuclear cells into a new tube. Wash the cells by re-suspending in 10 mL HBSS containing 2% FBS, 1% penicillin/streptomycin and 10 mM EDTA (isolation buffer), and then centrifuging at 4 oC 400 rcf for 5-10 min to pellet the cells. 
		Caution: Use only media that contains at least 1 g/L of glucose.</li>
	</ol>
	</li>
	<li>For CD11b positive selection, re-suspend 1 x 108 cells in 1 mL isolation buffer and incubate them for 15 min with 50 &#956;L of CD11b-conjugated magnetic beads at 4 oC.
	<ol>
		<li>Wash excess beads by adding 9 mL of isolation buffer and centrifuging at 400 rcf for 5-10 min at 4 oC. Aspirate the supernatants and re-suspend the cells in 1 mL isolation buffer. Apply the cells on a prewashed magnetic column according to manufacturer&#39;s instructions.</li>
		<li>Remove the column from the magnet, pipette 6 mL of isolation buffer onto the column, and flush out the magnetically-labeled cells into a sterile collection tube by pushing the plunger. Centrifuge cells at 400 rcf for 5-10 min at 4 oC. Wash the column twice with 3 mL of isolation buffer.</li>
		<li>Re-suspend cells at 1 x 107 cells/100 &#956;L isolation buffer and stain with the following fluorophore-conjugated antibodies: 0.1 &#956;g CD115, 0.25 &#956;g MHCII, and 0.1 &#956;g Ly-6C/1 x 106 cells.</li>
	</ol>
	</li>
	<li>Sort cells by gating on small side scatter (SSC) and small forward scatter (FSC) cells. 
	NOTE: Mouse inflammatory monocytes are generally defined as CD115+/MHCIIlo/neg/Ly6Chi, and patrolling monocytes are defined as CD115+/MHCIIlo/neg/Ly6Cneg 4,5. In cases where no separation between the two subsets is needed, total SSClo/FCSlo/CD115+/MHCIIlo cells can be sorted. Both na&#239;ve and tumor-bearing mice contain approximately 5 - 8 x 104 MoDC per 1 mL of blood and up to 4 - 5 x 105 MoDC following injection of GM-CSF. Of them, approximately 70% are inflammatory monocytes and 30% are patrolling monocytes.</li>
	<li>Plate the cells at a concentration of 1 x 106 cells/mL in a complete media supplemented with 20 ng/mL GM-CSF. After 1 day, transfer the non-adherent and loosely adherent cells to a new plate and culture for an additional 4-5 days. 
	Caution: DC media should not be supplemented with pyruvate.</li>
</ol>
3. Preparation of Tumor-IgG Immune Complexes
<ol>
	<li>Culture tumor cells in 75 cm2 culture flask to 70% confluence in complete DMEM media.
	<ol>
		<li>Add 2 mL of 0.25% trypsin/EDTA to detach cells from the culture flask and monitor cell morphology under a light microscope to avoid over-trypsinization.</li>
		<li>Add 8 mL of complete culture media (per 2 mL of trypsin) to inhibit trypsin digestion, and centrifuge at 4 oC, 400 rcf for 5-10 min to pellet the cells. 
		NOTE: Make sure to check tumor cells for Mycoplasma using a commercial PCR kit. Test for the presence of Gram-negative bacteria and fungal endotoxins using Limulus Amebocyte Lysate (LAL) assay28). Serum must be filtered through 0.22 &#956;M and tested for the 9 Code of Federal Regulations viruses. Additionally, test the culture media and serums by dropping 100 - 200 &#956;L onto LB agar or broth, and culture for 2 days at 37 oC.</li>
		<li>Wash cells again from trypsin and serum remains by re-suspending them in 10 mL PBS and centrifuge at 400 rcf for 5 - 10 min. Aspirate supernatant and repeat the wash 2 more times.</li>
	</ol>
	</li>
	<li>Fix cells in 1.8% buffered paraformaldehyde for 10 min at room temperature.
	<ol>
		<li>Wash the cells by re-suspending them in 10 mL PBS and centrifuge at 4 oC 400 rcf for 5 - 10 min.</li>
		<li>Aspirate supernatants and repeat wash two more times. 
		Optional: For tumor uptake assays, cells can be labeled by incubating them for 5 min at 37 oC in PBS containing 1 &#956;M carboxyfluorescein succinimidyl ester (CFSE). CFSE is then quenched with complete media for 10 min in ice. The cells should be washed extensively in PBS containing 2% serum to remove residual dye.</li>
	</ol>
	</li>
	<li>Re-suspend cells in FACS buffer (PBS supplemented with 2% FCS + 5 mM EDTA) and 0.5 &#956;g/mL of anti-CD16/32 in order to block potential non-specific protein-protein interactions. Plate in a U-shape 96 wells/plate at a concentration of 1 x 105 cells per 100 &#956;L.
	<ol>
		<li>Add different dilutions of tumor-binding antibodies ranging from 5 &#956;g - 5 ng/1 x 105 cells. Incubate the plate on ice for 15-20 min. 
		NOTE: IgG antibodies were isolated from the serum of na&#239;ve 20-24 weeks old female mice on protein A columns, as described24.</li>
	</ol>
	</li>
	<li>Wash cells by adding 150 &#956;L of PBS and centrifuging the plate at 4 oC 400 rcf for 5 - 10 min.
	<ol>
		<li>Discard the supernatants and repeat the wash twice. Re-suspend cells in 100 &#956;L FACS buffer containing fluorophore-conjugated secondary antibody. Incubate plate on ice for 20 min.</li>
		<li>Wash cells by adding 200 &#956;L of FACS buffer and centrifuging the plate at 400 rcf for 5 - 10 min. Discard the supernatants and repeat wash.</li>
		<li>Analyze tumor binding by flow cytometry and determine the minimal concentration required to coat the cells. 
		NOTE: Antibodies that do not increase mean fluorescence intensity (MFI) of stained tumor cells by at least fivefold over isotype control should not be used in subsequent functional assays.</li>
	</ol>
	</li>
</ol>
4. Activation of MoDC with Tumor-IgG IC
<ol>
	<li>Coat tumor cells with minimal IgG concentration, as described in sections 3.1 through 3.4.3
	<ol>
		<li>One day before activating MoDC with tumor IC, replace MoDC culture media, which contains GM-CSF. To do so, gently aspirate the media and wash the cells once with pre-warmed complete culture media. 
		NOTE: Isolated mature tumor-associated MoDC should be cultured for at least 2 - 3 h (or even overnight) after sorting in complete media without GM-CSF, and before activating them with IC.</li>
		<li>For tumor uptake analyses, add the CFSE-labeled tumor IC to MoDC at a ratio of 1:5 (IC:MoDC) and incubate overnight for 12 - 16 h in 1 mL of complete media per 1 x 106 DC.</li>
		<li>For FACS analyses of MoDC activation experiments, add tumor-IC at 1:1 (IC:MoDC) ratio and incubate overnight for 12 - 16 h. 
		NOTE: It is highly recommended to include a positive control well, in which MoDC are stimulated with 1 &#956;g/mL of LPS or other TLR agonists.</li>
		<li>Following overnight activation, aspirate the supernatants and wash cells gently three times with isolation buffer, or 10 mM EDTA HBSS.</li>
		<li>To detach DC from the plate, incubate cells for 2 - 3 min in 1 mL HBSS containing 10 mM EDTA, and detach cells by vigorous pipetting.</li>
		<li>Centrifuge cells at 400 rcf and re-suspend 1 x 106 DC in 90 &#956;L PBS supplemented with 2% FCS, 5 mM EDTA (FACS buffer) and 0.5 &#956;g of blocking antibodies. Incubate on ice for 5 - 10 min.</li>
		<li>Add 10 &#956;L of staining antibodies mixture to the cells and incubate on ice for 15 min.</li>
		<li>Add 2 mL FACS buffer to cells and centrifuge at 4 oC 400 rcf for 5 - 10 min.</li>
	</ol>
	</li>
	<li>Re-suspend cells in 200 &#956;L FACS buffer.
	<ol>
		<li>Add 0.5 - 1 &#956;g/mL of DAPI 1 - 2 min before running the samples, to exclude dead cells from analyses. Do not over-incubate DAPI, as MoDC will take it up within 10 - 15 min.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Limulus+amebocyte+lysate+(LAL)+to+LAL-reactive+glucans.">Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans.</a> J Clin Microbiol. 29 (11), 2477-2483 (1991).</li><li>Jahr, H., Pfeiffer, G., Hering, B. J., Federlin, K., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-mediated+activation+of+cytokine+production+in+human+PBMCs+by+collagenase+and+Ficoll.">Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll.</a> J Mol Med (Berl). 77 (1), 118-120 (1999).</li><li>Zhang, X., Morrison, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+selective+priming+effects+on+tumor+necrosis+factor+alpha+and+nitric+oxide+production+in+mouse+peritoneal+macrophages.">Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages.</a> J Exp Med. 177 (2), 511-516 (1993).</li><li>Hirohashi, N., Morrison, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+lipopolysaccharide+(LPS)+pretreatment+of+mouse+macrophages+modulates+LPS-dependent+interleukin-6+production+in+vitro.">Low-dose lipopolysaccharide (LPS) pretreatment of mouse macrophages modulates LPS-dependent interleukin-6 production in vitro.</a> Infect Immun. 64 (3), 1011-1015 (1996).</li><li>Deng, H., Maitra, U., Morris, M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+responsible+for+the+priming+of+macrophage+activation.">Molecular mechanism responsible for the priming of macrophage activation.</a> J Biol Chem. 288 (6), 3897-3906 (2013).</li><li>Cella, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+inhibitory+receptor+(ILT3)+expressed+on+monocytes,+macrophages,+and+dendritic+cells+involved+in+antigen+processing.">A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing.</a> J Exp Med. 185 (10), 1743-1751 (1997).</li><li>Kramer, P. R., Winger, V., Reuben, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+limits+TNF-alpha+production+in+CD16-activated+monocytes.">PI3K limits TNF-alpha production in CD16-activated monocytes.</a> Eur J Immunol. 39 (2), 561-570 (2009).</li><li>Rose, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+gamma+receptor+cross-linking+activates+p42,+p38,+and+JNK/SAPK+mitogen-activated+protein+kinases+in+murine+macrophages:+role+for+p42MAPK+in+Fc+gamma+receptor-stimulated+TNF-alpha+synthesis.">Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.</a> J Immunol. 158 (7), 3433-3438 (1997).</li><li>Rezzonico, R., Imbert, V., Chicheportiche, R., Dayer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation+of+CD11b+and+CD11c+beta(2)+integrins+by+antibodies+or+soluble+CD23+induces+macrophage+inflammatory+protein+1alpha+(MIP-1alpha)+and+MIP-1beta+production+in+primary+human+monocytes+through+a+pathway+dependent+on+nuclear+factor-kappaB.">Ligation of CD11b and CD11c beta(2) integrins by antibodies or soluble CD23 induces macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta production in primary human monocytes through a pathway dependent on nuclear factor-kappaB.</a> Blood. 97 (10), 2932-2940 (2001).</li></ol>

All authors declare they have no conflict of interest and that they have nothing to disclose.

Given the large number of DC required for vaccinating mice (approximately 2-4 x 106 DC per one mouse), most of the vaccination strategies in mice are based on isolation of DC from BM and spleen followed by their ex vivo activation. However, attempts to activate tumor DC in vivo, using the same conditions for activating spleen and BM DC, have often been unsuccessful in producing effective immunity. In two subsequent publications, Carmi et al. have found that blood and tumor MoDC diffe...

Since their discovery, dendritic cells (DC) have been a focus of extensive research due to their unique ability to skew T cell differentiation1. Over the past several decades, an extensive research effort has sought to define the various DC subsets and their function during tumor progression and immunity 2. DCs are composed of heterogeneous cell populations that differ from each other in their pattern-recognition receptors, tissue distribution, and migratory and antigen presentation capabilities3,4,5. Compared to other DC subsets, monocyte-derived DC (MoDC) are far more abundant in tumors and can be easily generated from circulating or tumor-infiltrating monocytes6,7. Therefore, many clinical trials seeking to take advantage of their relative prevalence are based on in vivo and ex vivo manipulation of autologous MoDC in order to elicit T cell immunity 8,9.
Similarly, DC-based vaccination of experimental tumor models requires 2-3 serial injections, 5-7 days apart, of 1-2 x 106 activated DC pulsed with tumor antigens. Therefore, to achieve this large number of DC, most mouse studies have primarily used MoDC cultured from bone marrow (BM) precursors in GM-CSF for 7-9 days (IL-4 is not needed in the mouse setting)10,11. Nonetheless, given that GM-CSF knockout mice have overall normal DC compartment 12,13, and given the mixed populations obtained from that culture,14 the physiological relevance of these DC has been called into question.
Alternatively, DC may be routinely isolated from spleen cells. However, DC comprise only about 0.3-0.8% of total spleen cells (resulting in approximately 7 x 105 DC/spleen), and of these cells, only CD103+ DC and MoDC can migrate back to lymphoid organs. Since MoDCs comprise approximately 10-15% of splenic DC populations15,16, most isolation protocols yield approximately 1 x 105 MoDC per spleen. Expansion of MoDC can be achieved by injecting transfected B16 cells that secrete GM-CSF, resulting in a 100-fold increase in splenic MoDC17. However, the use of MoDC for developing DC vaccines is limited since this procedure cannot be done in humans and the obtained MoDC are already highly activated.
In addition to obtaining adequate numbers of DC, another challenge for developing effective DC vaccines against autologous cancer cells involves the lack of sufficient danger signals in the tumor setting to fully activate DC. Induction of co-stimulatory signals is usually achieved through activation of pattern-recognition receptors (PRR), or c-type lectin signaling pathways18,19,20,21. A further approach for activating DC exploits their ability to take up antigens through interactions with surface Fc&#947; receptors (Fc&#947;R). Indeed, a number of important manuscripts have shown that injection of MoDC from BM precursors activated with tumor-IgG IC can prevent tumor growth in prophylactic settings, and can lead to the eradication of established tumors22,23.
In two recent papers, Carmi et al. discovered that in contrast to BMDC and spleen DC, MoDC from the blood and tumors cannot respond to IgG IC without additional stimuli. This was found to be due to the presence of high intracellular levels of tyrosine phosphatases regulating Fc&#947;R signaling24,25. By defining a critical checkpoint in DC, this work provided an important insight into the requirements for successful DC-based vaccination. The requirement for additional stimuli to enable Fc&#947;R signaling, and presumably signaling from other lectin receptors utilizing a similar phosphorylation cascade, thus underscores the need for avoiding the priming of DC during their isolation.
Therefore, the present protocol describes the isolation of MoDC from blood and tumors, which differ markedly from BM and spleen DC, and highlights precautions worth considering during the process.

<table><tbody><tr><td>Ficoll-Paque PREMIUM</td><td>GE-Healthcare</td><td>17-5442-02</td><td></td></tr><tr><td>OptiPrep</td><td>StemCell Technologies</td><td>07820</td><td></td></tr><tr><td>CD45 MicroBeads</td><td>Miltenyi</td><td>130-052-301</td><td></td></tr><tr><td>EasySep Monocyte Isolation Kit</td><td>StemCell Technologies</td><td>19861</td><td></td></tr><tr><td>Collagenase IV</td><td>Sigma</td><td>C9697-50MG</td><td>Test each lot for endotoxin</td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25-10MG</td><td></td></tr><tr><td>HBSS</td><td>ThermoFisher</td><td>14025092</td><td></td></tr><tr><td>FBS</td><td>ThermoFisher</td><td>16140071</td><td>Test each lot for endotoxin</td></tr><tr><td>PE-CD11c</td><td>Biolegend</td><td>117307</td><td></td></tr><tr><td>APC-CD11b</td><td>Biolegend</td><td>101211</td><td></td></tr><tr><td>Brilliant Violet 650 MHCII</td><td>Biolegend</td><td>107641</td><td></td></tr><tr><td>AF48- CD86</td><td>Biolegend</td><td>105017</td><td></td></tr><tr><td>APC/Cy7-Ly-C6</td><td>Biolegend</td><td>108423</td><td></td></tr><tr><td>PE/Cy7-CD15</td><td>Biolegend</td><td>135523</td><td></td></tr></tbody></table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

We initially compared the capacity of antibodies from na&#239;ve syngeneic and allogeneic mice to bind to tumor cells. To this end, B16F10 and LMP tumor cell lines were fixed in paraformaldehyde and washed extensively. B16F10 is a melanoma cell line, which was originally isolated from lung metastases in C57Bl/6 mice. LMP is a pancreatic tumor cell that was isolated from KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre mice, and grows steadily in 129F1 mice. To obtain IC, tumor cells were incub...

Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

isolation-protocol-mouse-monocyte-derived-dendritic-cells-their

Research

JoVE Journal

Cancer Research

11.3K Views.  Tel-Aviv University. Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Video: Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Immunology and Infection

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Biology

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

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Isolation of Mouse Lung Dendritic Cells

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Isolation and Activation of Murine Lymphocytes

An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Isolation of Immune Cells from Primary Tumors

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14⁺ Monocytes